Project Summary/Abstract Changes in dietary and exercise patterns have led to a pandemic of the metabolic syndrome, which is characterized by visceral obesity, insulin resistance, dyslipidemia, and a strong predisposition to atherosclerosis. Increased expression of plasminogen activator inhibitor-1 (PAI-1) is also a hallmark of the metabolic syndrome. PAI-1 is a serine protease inhibitor that rapidly inhibits tissue- and urokinase-type plasminogen activators, thereby stabilizing fibrin and promoting thrombosis. PAI-1 is also an important mediator of insulin resistance and cellular processes that drive atherosclerosis development, including vascular cell migration and senescence. Our group has shown that PAI-039, a specific inhibitor of PAI-1, produces significant anti-atherosclerosis, anti-obesity, and anti-inflammatory effects in a murine model of the metabolic syndrome. This work is the first to demonstrate that drug targeting of the fibrinolytic system inhibits atherosclerosis formation. The objectives of this proposal are to define the mechanisms by which PAI-1 inhibitors produce these beneficial effects and determine their safety and efficacy under clinically relevant conditions. It is hypothesized that pharmacological targeting of PAI-1 will 1) inhibit atherosclerosis formation by direct vascular effects, including inhibition of macrophage invasion into plaques, vascular cell senescence, and cholesterol uptake into the arterial wall, 2) inhibit obesity by increasing energy expenditure, uncoupling protein (UCP) expression, and beige adipocyte formation in adipose tissue, and 3) attenuate insulin resistance and pathological fluxes of free fatty acids and cholesterol in the metabolic syndrome. An interdisciplinary team of investigators with extensive experience in vascular biology, obesity, glucose and lipid metabolism, and the biochemistry and pharmacology of PAI-1 has been assembled to carry out this work. The proposed studies will involve PAI-039 and another novel PAI-1 inhibitor, CDE-268. These compounds will be studied in murine models of the metabolic syndrome, as well as in lean mice, using a series of novel and sophisticated methods aimed at defining the mechanisms by which drug targeting of PAI-1 inhibits atherosclerosis, adipose tissue dysfunction, insulin resistance, and dyslipidemia. Ossabaw swine, a highly translational model of the metabolic syndrome and atherosclerosis formation, will also be employed, thus leveraging the outstanding environment at the University of Missouri for studying large animal models of human disease. These studies will determine the efficacy and safety of long-term administration of PAI-1 inhibitors under clinically relevant conditions. The central premise of this proposal is that PAI-1 is a drug target for treating metabolic syndrome and reducing cardiovascular risk. This work has major public health implications, as obesity and atherosclerosis are amongst the most important diseases facing US citizens. Successful completion of these studies will define the pathological functions of PAI-1 in obesity and atherosclerosis and position our team to initiate a phase I clinical trial of pharmacologic PAI-1 inhibition in humans with metabolic syndrome.